The following discussion of the background to the invention is intended to facilitate an understanding of the present invention. However, it should be appreciated that the discussion is not an acknowledgment or admission that any of the material referred to was published, known or part of the common general knowledge in any jurisdiction as at the priority date of the application.
Birth defects are one of the leading causes of infant mortality worldwide1,2,3 and can also result in long-term disabilities and illnesses. Abnormalities during fetal development can be due to either genetic conditions or environmental exposure during pregnancy, especially the first trimester. Therefore, regulatory bodies, such as the US Environmental Protection Agency (EPA), have mandated that environmental agents, such as drugs, chemicals and pesticides should be evaluated for developmental toxicity4.
Testing developmental toxicity on animals is limited by cost and ethical issues. Hence, a number of alternative animal embryo or cell-based in vitro developmental models have been developed, which include the frog embryo teratogenesis assay (FETAX)5, the chicken embryo toxicity screening test (CHEST)6, the micromass (MM) assay using mouse embryonic mesenchymal cells7, the mouse or rat whole embryo culture (WEC) assay8, the zebrafish embryo-larva developmental toxicity assay9, and the mouse embryonic stem cell test (EST)10.
According to the European Centre for the Validation of Alternative Methods (ECVAM), only the MM assay, WEC and mouse EST for embryotoxicity testing are scientifically validated and can be considered for regulatory acceptance and application11-13. However, there are inter-species variations that cannot adequately explain differences in the molecular regulation of embryonic development between human and animals, or when used in reproductive toxicity testing, generate false negatives with devastating consequences. For instance, the withdrawn drug, Thalidomide, causes developmental deformation in human but not in mouse14. This technology aims to provide a human cell-based developmental model for research and screening applications in developmental toxicity.
There have been attempts to replace mouse ESCs used in the EST assay with human embryonic stem cells (hESCs)16,17, although there have been no successful development of a human EST. The core standard of mouse EST is to evaluate the toxicity of compounds based on their effect on mouse embryonic stem cell (mESC) differentiation to beating cardiomyocytes10. By using mouse ES cell line D3 and mouse embryonic fibroblast cell line 3T3, they try to measure inhibition of cytotoxicity (IC50) values for both cell lines, and inhibition of differentiation (ID50) values for mESCs. They further classify the drug compounds into three classes as “non-embryotoxic”, “weakly embryotoxic” and “strongly embryotoxic” using validated prediction model. The entire process lasts for 10 days using traditional beating cardiomyocytes monitoring under microscope or 7 days using FACS to check the gene expression of cardiac tissue. One major reason is that hESCs differentiate in vitro into cardiomyocytes at a slower rate and take longer time than mESCs (10-25% after 30 days)18, making it impossible to count the beating cardiomyocytes on Day 10. Using RT-PCR or immunostaining method, researchers could acquire the data describing the effects of developmental toxins. However, lacking suitable scoring system makes it still unsatisfactory for drug testing application.
In 2010, Cezar's group showed for the first time the successful classification of drugs into developmental toxins and non-developmental toxins using metabolomics and random forest modeling19. However, they just tested those drugs in hESC pluripotency maintenance medium (i.e., mTeSR1 medium) instead of actually differentiating hESC, and they cannot further classify those developmental toxins into weak or strong developmental toxic compounds since only circulating concentration of the drugs was applied in the experiments.
While the current cell-based MM and EST assays can potentially incorporate human embryonic or pluripotent cells, the developmental process (i.e. cells differentiating into the 3 germ layers) is spontaneous, and disorganized in these models. Consequently, they do not provide a sensitive and reliable way of classifying developmental toxins, which includes embryo toxins and teratogens, because the assays are either measuring general cytotoxicity7 or inhibition of cardiac tissue formation10, and are too crude to capture an important aspect of developmental toxicity—disruption of differentiation patterns. Current EST models rely on measuring inhibition of cardiomyocyte formation by xenobiotics as an indicator of developmental toxicity. Therefore it is not compatible with the intrinsic property of hESCs as hESCs do not form cardiomyocytes readily, unlike mouse ES cells.
The object of the invention is to ameliorate at least one of the problems mentioned herein.